CN110308283B - Laminin calibrator and quality control product - Google Patents
Laminin calibrator and quality control product Download PDFInfo
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- CN110308283B CN110308283B CN201910572406.XA CN201910572406A CN110308283B CN 110308283 B CN110308283 B CN 110308283B CN 201910572406 A CN201910572406 A CN 201910572406A CN 110308283 B CN110308283 B CN 110308283B
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Abstract
The invention relates to a laminin calibrator and a quality control product, wherein the laminin calibrator comprises an antigen, a buffer solution, a preservative and urea; the concentration of the urea is 0.05-0.2 mol/L; the laminin quality control product comprises antigen, serum, preservative and urea; the concentration of the urea is 0.05-0.2 mol/L. According to the laminin calibration product and the laminin quality control product provided by the invention, urea plays a certain role in protecting the laminin stability after being added, so that the stability of both the calibration product and the quality control product is improved. The invention fills the blank in the field of laminin calibration products and quality control products, and also plays a guiding role in the development of urea used for other similar calibration products and quality control products.
Description
Technical Field
The invention belongs to the technical field of immunodiagnosis, and particularly relates to a stable and uniform laminin calibration material and a quality control material prepared by adding urea as a protein protective agent.
Background
Hepatic fibrosis is a pathophysiological process, which refers to abnormal proliferation of connective tissue in the liver caused by various pathogenic factors. Any liver injury has liver fibrosis in the process of liver repair and healing, and if the injury factor cannot be removed for a long time, the fibrosis process can be continuously developed into liver cirrhosis for a long time. Many chronic liver diseases can cause liver fibrosis.
The existing methods for diagnosing liver fibrosis comprise liver histopathology examination, liver ultrasonic testing and serology methods, although the pathology examination is used as a gold standard for liver fibrosis diagnosis, certain defects exist, wounds exist, potential complications exist, and the methods are not easy to be accepted by patients, and the method cannot monitor the disease development and the curative effect of liver fibrosis. Liver ultrasound tests solve the drawbacks of pathological examination methods, but the methods are not easy to succeed for overweight and obese patients, and are also affected by indices such as large blood vessels in the liver, cholestasis, ascites, and the like. The in vitro diagnosis serology method well solves the limitations of the 2 detection methods, is rapid, accurate and real-time, and is the development direction of the future hepatic fibrosis diagnosis method. Items for serological detection of liver fibrosis are generally 4, type iii procollagen, type iv collagen, laminin and hyaluronic acid. At present, the number of commercialized kits is small, related literature reports are few, the field is new, the commercial prospect is optimistic, and the medical research and commercial value is high.
Laminin was discovered in 1979 as a heterotrimer composed of α, β, and γ 3 polypeptide chains, a macromolecular glycoprotein. These alpha, beta and gamma chains can be assembled into ten or T-shaped ten different laminins, and then these peptide chains are coiled into alpha-helical structure, so that the rod-shaped portion and ball-shaped portion appear on laminin structure, and these structures mostly can be retained by means of hydrogen bond, but in aqueous solution the laminin structure is susceptible to other factors, so that the protein structure can be damaged.
Urea is a polar molecule of important biological function and one of the most commonly used protein denaturants. In the urea-water mixed solvent, urea molecules are accumulated on the surface of protein molecules instead of water molecules, the direct interaction between the urea molecules and the protein molecules has complex influence on the conformation of the protein, the high-concentration urea-water mixed solvent destroys the conformation of the protein, and the low-concentration mixed solvent is favorable for the protein to form a tighter conformation.
Although urea is a denaturant and has no related application in the field of immunodiagnosis at present, the application of the urea substance to the coating of magnetic beads or the labeling of luminescent substrates in the research and development process of the company does not obtain good effect, and the urea substance can have a polar effect to cause the coating or labeling process, and the protein is not stably combined with the magnetic beads or the luminescent substrates. However, depending on the effect of urea on protein conformation, urea is applied to substances that do not require ligation or other treatment, such as urea in a protein protecting solution, in an attempt to protect the protein.
Therefore, the urea is applied to the laminin calibrator and the laminin quality control product, the appropriate urea concentration is adjusted, the protection effect of the urea on laminin in the aqueous solution is researched, the stable calibrator and the stable quality control product can be obtained, and the guidance and reference effects on improving the stability of other similar proteins can be realized.
Disclosure of Invention
The invention aims to provide a stable and uniform laminin calibration material and a quality control material prepared by adding urea as a protein protective agent.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention provides a laminin calibrator, which comprises an antigen, a buffer solution and a preservative;
also comprises urea;
the concentration of the urea is 0.05-0.2 mol/L.
In the technical scheme, the laminin calibration product further comprises a salt substance, a polysaccharide substance and purified water.
In the technical scheme, the laminin calibration product comprises the following components in percentage by weight:
in the above technical scheme, the pH of the laminin calibrator is 6.5-7.5.
In the above technical scheme, the buffer solution is PB or MPOSO, the salt substance is sodium chloride, the polysaccharide substance is sucrose or trehalose, and the preservative is P300 or sodium azide.
The invention also provides a laminin quality control product, which comprises an antigen, serum and a preservative;
also comprises urea;
the concentration of the urea is 0.05-0.2 mol/L.
In the technical scheme, the laminin quality control product further comprises a salt substance and a polysaccharide substance.
In the technical scheme, the laminin quality control product comprises the following components in percentage by weight:
in the technical scheme, the pH value of the laminin quality control product is 6.5-7.5.
In the above technical scheme, the serum is animal serum, human serum or similar serum, preferably bovine serum, the salt substance is sodium chloride, the polysaccharide substance is sucrose or trehalose, and the preservative is P300 or sodium azide.
The invention has the beneficial effects that:
according to the laminin calibration product and the laminin quality control product provided by the invention, urea plays a certain role in protecting the laminin stability after being added, so that the stability of both the calibration product and the quality control product is improved. The invention fills the blank in the field of laminin calibration products and quality control products, and also plays a guiding role in the development of urea used for other similar calibration products and quality control products.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a graph showing the unsealing stability of laminin calibration products and quality control products provided by the present invention.
FIG. 2 is a graph showing the real-time stability of laminin calibration product and quality control product provided by the present invention.
Detailed Description
The invention provides a laminin calibrator, which comprises an antigen, a buffer solution and a preservative; also comprises urea; the concentration of the urea is 0.05-0.2 mol/L.
Preferably, the laminin calibration product further comprises a salt substance, a polysaccharide substance and purified water.
Further preferably, the laminin calibration product comprises the following components in percentage by weight:
the laminin calibrator has a pH of 6.5-7.5.
Preferably, the buffer solution is PB or MPOSO, the salt substance is sodium chloride, the polysaccharide substance is sucrose or trehalose, and the preservative is P300 or sodium azide.
The invention also provides a laminin quality control product, which comprises an antigen, serum and a preservative; also comprises urea; the concentration of the urea is 0.05-0.2 mol/L.
Preferably, the laminin quality control product also comprises a salt substance and a polysaccharide substance.
Further preferably, the components of the laminin quality control product and the concentrations of the components are as follows:
the pH value of the laminin quality control product is 6.5-7.5.
Preferably, the serum is animal serum, human serum or similar serum, more preferably bovine serum, the salt substance is sodium chloride, the polysaccharide substance is sucrose or trehalose, and the preservative is P300 or sodium azide.
The technical scheme of the invention is clearly and specifically described in the following by combining specific embodiments.
The following examples and comparative examples use equipment details of 10 ten thousand electronic balance, pH meter and associated calibration buffer, 1000mL graduated cylinder and special filter.
Example 1
The laminin calibrator provided by the invention comprises the following components in percentage by weight:
according to the concentration ratio: adding a full batch of purified water into a clean preparation container, sequentially adding a full batch of buffer solution, salt substances, polysaccharide substances, urea and preservative into the purified water in sequence, and sequentially stirring the mixture until the buffer solution, the salt substances, the polysaccharide substances, the urea and the preservative are completely dissolved; filtration was performed once using a 0.22 μm filter. The pH was adjusted to 7.0 (25. + -. 1 ℃ C.) with concentrated HCl or 40mmol/LNaOH, laminin was added in concentration such that the concentrations of calibrators were low: 100 ng/mL; high value: 500 ng/mL. Subpackaging according to the specification of 1 mL/bottle.
Comparative example 1
The difference from example 1 is that the concentration of urea is 0, 0.02, 0.5, 2 mol/L.
Example 2
Adding a whole batch of serum into a clean preparation container according to the proportion, sequentially adding a whole batch of salt substances, polysaccharide substances, urea and preservatives into the serum, and sequentially stirring until the salt substances, the polysaccharide substances, the urea and the preservatives are completely dissolved; filtration was performed once using a 0.22 μm filter. The pH was adjusted to 7.0 (25. + -. 1 ℃ C.) with concentrated HCl or 40mmol/LNaOH, and laminin was added in concentration such that the concentrations of the quality control were level 1: 100 ng/mL; level 2: 500 ng/mL. Subpackaging according to the specification of 1 mL/bottle.
Comparative example 2
The difference from example 2 is that the concentration of urea is 0, 0.02, 0.5, 2mol/L, respectively.
The calibrator and the quality control prepared in the above examples and comparative examples were stored at 2-8 ℃ or 37 ℃ for 14 days, and then the laminin assay kit (chemiluminescence immunoassay) was selected and the effect of urea of different concentrations on the stability of laminin calibrator and quality control was tested using a CM-180 full-automatic chemiluminescence immunoassay analyzer (table 1, table 2).
Table 1 accelerated stability of laminin calibrators at different concentrations of urea for 14 days
Note: calibrator concentration units ng/mL.
TABLE 2 14-day accelerated stability of laminin quality control products at different urea concentrations
Note: the concentration unit of the quality control material is ng/mL.
As can be seen from the above table, after urea is added, the accelerated stability attenuation rate of the laminin calibration product and the quality control product is improved, the laminin is damaged when the concentration of urea is too high, the laminin is not protected when the concentration of urea is too low, and the laminin is only protected when the concentration of urea is within the range of 0.05-0.2 mol/L.
After the content of the urea is determined, a calibrator and a quality control material with the concentration of 0.05mol/L urea are selected for unsealing stability (figure 1), real-time stability (figure 2) and uniformity index verification (table 3), so that the urea is determined to be capable of improving the stability of the laminin calibrator and the quality control material, and the urea has stable and uniform performance and can be applied to a matched reagent as a matched calibrator and a matched quality control material of a reagent.
TABLE 3 homogeneity test data for laminin calibrator and quality control product
Note: concentration unit ng/mL of calibrator and quality control material
The urea is added into a laminin calibration product and a laminin quality control product, and only proper urea concentration plays a role in protecting laminin.
Example 3
The laminin calibrator provided by the invention comprises the following components in percentage by weight:
according to the concentration ratio: adding a full batch of purified water into a clean preparation container, sequentially adding a full batch of buffer solution, salt substances, polysaccharide substances, urea and preservative into the purified water in sequence, and sequentially stirring the mixture until the buffer solution, the salt substances, the polysaccharide substances, the urea and the preservative are completely dissolved; filtration was performed once using a 0.22 μm filter. The pH was adjusted to 6.5 (25. + -. 1 ℃ C.) with concentrated HCl or 40mmol/LNaOH, laminin was added in concentration such that the concentrations of calibrators were low: 100 ng/mL; high value: 500 ng/mL. Subpackaging according to the specification of 1 mL/bottle.
This example prepares a stable, uniform laminin calibrator by adding urea as a protein protectant.
Example 4
The laminin calibrator provided by the invention comprises the following components in percentage by weight:
according to the concentration ratio: adding a full batch of purified water into a clean preparation container, sequentially adding a full batch of buffer solution, salt substances, polysaccharide substances, urea and preservative into the purified water in sequence, and sequentially stirring the mixture until the buffer solution, the salt substances, the polysaccharide substances, the urea and the preservative are completely dissolved; filtration was performed once using a 0.22 μm filter. The pH was adjusted to 7.5 (25. + -. 1 ℃ C.) with concentrated HCl or 40mmol/LNaOH, laminin was added in concentration such that the concentrations of calibrators were low: 100 ng/mL; high value: 500 ng/mL. Subpackaging according to the specification of 1 mL/bottle.
This example prepares a stable, uniform laminin calibrator by adding urea as a protein protectant.
Example 5
Adding a whole batch of serum into a clean preparation container according to the proportion, sequentially adding a whole batch of salt substances, polysaccharide substances, urea and preservatives into the serum, and sequentially stirring until the salt substances, the polysaccharide substances, the urea and the preservatives are completely dissolved; filtration was performed once using a 0.22 μm filter. The pH was adjusted to 6.5 (25. + -. 1 ℃ C.) with concentrated HCl or 40mmol/LNaOH, laminin was added in concentration such that the concentrations of the quality control were level 1: 100 ng/mL; level 2: 500 ng/mL. Subpackaging according to the specification of 1 mL/bottle.
This example prepares a stable, uniform laminin quality control by adding urea as a protein protectant.
Example 6
Adding a whole batch of serum into a clean preparation container according to the proportion, sequentially adding a whole batch of salt substances, polysaccharide substances, urea and preservatives into the serum, and sequentially stirring until the salt substances, the polysaccharide substances, the urea and the preservatives are completely dissolved; filtration was performed once using a 0.22 μm filter. The pH was adjusted to 7.5 (25. + -. 1 ℃ C.) with concentrated HCl or 40mmol/LNaOH, laminin was added in concentration such that the concentrations of the quality control were level 1: 100 ng/mL; level 2: 500 ng/mL. Subpackaging according to the specification of 1 mL/bottle.
This example prepares a stable, uniform laminin quality control by adding urea as a protein protectant.
Example 7
Adding a whole batch of serum into a clean preparation container according to the proportion, sequentially adding a whole batch of salt substances, polysaccharide substances, urea and preservatives into the serum, and sequentially stirring until the salt substances, the polysaccharide substances, the urea and the preservatives are completely dissolved; filtration was performed once using a 0.22 μm filter. The pH was adjusted to 7.0 (25. + -. 1 ℃ C.) with concentrated HCl or 40mmol/LNaOH, and laminin was added in concentration such that the concentrations of the quality control were level 1: 100 ng/mL; level 2: 500 ng/mL. Subpackaging according to the specification of 1 mL/bottle.
This example prepares a stable, uniform laminin quality control by adding urea as a protein protectant.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (2)
1. A laminin calibrator is characterized by comprising the following components in percentage by weight:
the pH of the laminin calibrator is 6.5-7.5;
the buffer solution is PB or MPOSO, the salt substance is sodium chloride, the polysaccharide substance is sucrose or trehalose, and the preservative is P300 or sodium azide;
2. a laminin quality control product is characterized by comprising the following components in percentage by weight:
the pH value of the laminin quality control product is 6.5-7.5;
the serum is animal serum, human serum or quasi-serum, the salt substance is sodium chloride, the polysaccharide substance is sucrose or trehalose, and the preservative is P300 or sodium azide.
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